Specific Aim 1: Creating human monoclonal antibodies. Antibody engineering is typically carried out by displaying human antibodies or antibody fragments on the surface of microorganisms (e.g. phage, bacteria and yeast). By taking advantage of highly efficient protein expression in human HEK-293T cells and high throughput screening features of flow cytometry, we have developed a new method called mammalian cell display. Using this approach, functional single-chain antibody variable fragments (scFvs) are expressed in human HEK-293 cells and high affinity antigen binders are subsequently identified and isolated from a combinatory library via flow cytometry. HEK 293 cells are more likely than microorganisms to correctly express antibodies. We and others have shown that mammalian cell display can also be used to mimic somatic hypermutation for antibody affinity maturation by targeting intrinsic hotspots or co-expressing activation-induced cytidine deaminase (AID), the key enzyme for somatic hypermutation in B cells. In FY09-10, our work on mammalian cell display resulted in two method papers published in Methods in Molecular Biology and in several presentations at national and international therapeutic antibody conferences. Due to their lower immunogenicity in patients, a fully human monoclonal antibody (mAb) is the most desirable antibody format for clinical application. Mesothelin is a promising new therapeutic target given its high expression in a variety of cancers including mesothelioma and ovarian cancer. Previous research by Ira Pastan and colleagues led to the development of an anti-mesothelin immunotoxin (SS1P). It contains a murine SS1 Fv fused to a bacterial toxin. A chimeric antibody (MORAb-009) containing the same murine Fv was also developed and is currently being examined in a Phase II clinical trial for mesothelioma and pancreatic cancer. In FY08-09, we (1) identified HN1 as a high affinity Fv specific for mesothelin, (2) produced a fully human IgG molecule based on the HN1 Fv, and (3) showed that HN1 was able to bind specifically to cell surface-associated mesothelin on mesothelioma and ovarian cancer cells with high affinity. This work yielded a patent application. In FY10, we continued to characterize HN1 and demonstrated it could specifically kill cancer cells with strong antibody-dependent cell-mediated cytotoxicity (ADCC). In FY10, our work on HN1 was published in the International Journal of Cancer and presented at several international cancer and antibody meetings. This work was in part supported by the Mesothelioma Applied Research Foundation and the Ovarian Cancer Research Fund. In FY09, we became interested in glypican-3 (GPC3) as a new liver cancer-specific antigen. GPC3 is a promising candidate for liver cancer therapy given that it is not expressed in normal tissues and shows high expression in hepatocellular carcinoma (HCC). Loss-of-function mutations of GPC3 cause Simpson-Golabi-Behmel syndrome, a rare X-linked overgrowth disorder. GPC3 binds, through its N-terminus, to IGF-2 and IGF-1R, and through its C-terminus to Wnt and Hedgehog proteins. GPC3 also binds FGF-2 through its heparan sulfate glycan chains. In FY10, we made recombinant soluble GPC3 (sGPC3) protein in HEK-293 cells and found that sGPC3 protein could inhibit the proliferation of HCC cells. This work yielded a publication in the International Journal of Cancer and a patent application in FY10. Our ongoing studies are focused on the development of novel mAbs targeting GPC3 to inhibit HCC growth. Specific Aim 2: Designing novel immunoconjugates. Ovarian cancer and mesothelioma frequently express both mesothelin and mucin CA125/MUC16. The interaction between mesothelin and CA125/MUC16 may facilitate the implantation and peritoneal spread of tumors by cell adhesion. However, the detailed nature of this molecular interaction is poorly understood. In FY09, we identified a novel distinct functional binding domain (called IAB) in mesothelin for CA125/MUC16. We further showed that IAB could effectively block the binding of mesothelin to CA125 and inhibit cancer cell adhesion by a novel mechanism. IAB is a good candidate for use as an antagonist to treat CA125-expressing tumors. Our work targeting CA125 yielded one publication and an international patent application in FY09. Isolation of a human mAb is not always possible, especially for those antigens with low immunogenicity in humans. One such antigen is CA125. Since the generation of OC125 in 1981, numerous mouse mAbs directed against CA125 have been developed. A mouse mAb called B43.13 (also called Oregovomab) was first developed as a tumor-imaging agent to detect recurrent ovarian cancer and has been evaluated in Phase II clinical studies for ovarian cancer treatment. However, a fully human mAb against CA125 has not been described, possibly due to the role of CA125 in down-regulation of the immune system. In FY10, we made the IAB-human Fc hybrid molecule called immunoadhesin HN125 and showed it could bind CA125-expressing cancer cells with high affinity and functionally block the CA125-mesothelin interaction. We are currently examining its anti-tumor activity.